Double-stranded oligonucleotides, conjugates, compositions, and uses targeting the INHBE gene

Abstract

The present invention discloses double-stranded oligonucleotides, conjugates, and compositions targeting the INHBE gene, and relates to the technology of nucleic acid drugs. The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand and the sense strand are complementary or nearly complementary, and the sense strand comprises a nucleotide sequence that is the same as or nearly identical to at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence, where nearly identical means that there is a difference of three or fewer nucleotides between the sense strand and at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence. The double-stranded oligonucleotide can effectively suppress the INHBE gene and treat patients suffering from metabolic disorders or metabolic syndrome, and related diseases such as diabetes, hypertension, and cardiovascular disease.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to a Chinese patent application filed with the China Patent Administration on August 21, 2023, with application number 202311053101.0, and with the title of invention "Double-stranded oligonucleotides, conjugates, compositions and their use targeting the INHBE gene," the entire contents of which are incorporated herein by reference.

[0002] This disclosure belongs to the field of nucleic acid pharmaceuticals, and in particular relates to double-stranded oligonucleotides targeting the INHBE gene, conjugates of said double-stranded oligonucleotides, and includes pharmaceutical compositions of said double-stranded oligonucleotides and / or double-stranded oligonucleotide conjugates and their use. [Background technology]

[0003] Currently, metabolic disorders are common, and conventional medical treatments include lipid-lowering agents such as statins and other medications. However, these treatments are usually limited by frequent dosing and drug interactions. There are currently no commercially available drugs that can effectively silence metabolic disorder-related genes, such as drugs that suppress INHBE to further effectively suppress metabolic disorders.

[0004] Therefore, in this field, there is a strong need for drugs that can effectively suppress targeted INHBE genes to treat subjects suffering from metabolic disorders or metabolic syndrome and related diseases such as diabetes, hypertension, and cardiovascular disease. [Overview of the Initiative]

[0005] In view of this, the present disclosure provides double-stranded oligonucleotides, conjugates, compositions, and uses thereof that target the INHBE gene. The present disclosure describes how to treat diseases or conditions associated with the INHBE gene by suppressing the expression of the INHBE gene by administering an INHBE-specific inhibitor (double-stranded oligonucleotide, conjugate, composition, etc.) to a subject.

[0006] To solve the above technical problems, this disclosure proposes the following technical solutions.

[0007] In one aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide targeting the INHBE gene, comprising a sense strand and an antisense strand, wherein the antisense strand and the sense strand are complementary or nearly complementary, with nearly complementary meaning that there are three or fewer nucleotide mismatches in the double-stranded regions of the sense strand and the antisense strand.

[0008] Furthermore, the double-stranded oligonucleotide comprises one sense strand and one antisense strand, the antisense strand comprising at least 15 consecutive nucleotides in any of the sequences indicated by SEQ ID NO:155 to SEQ ID NO:308 in Table 1, or a nucleotide sequence in which the difference from the at least 15 consecutive nucleotides is 3 or less.

[0009] The sense strand includes a nucleotide sequence that forms a double-stranded region at least partially inversely complementary or nearly complementary with the antisense strand, where nearly complementary means that there are three or fewer nucleotide mismatches in the double-stranded regions of the sense strand and the antisense strand.

[0010] In some preferred embodiments of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0011] In aspect 2 of the present disclosure, the present disclosure provides a double-stranded oligonucleotide conjugate comprising the double-stranded oligonucleotide and a ligand capable of binding to one or more cell receptors. A double-stranded oligonucleotide conjugate is provided.

[0012] In some preferred embodiments of the present disclosure, the ligand is conjugated to the sense strand and / or the antisense strand.

[0013] In aspect 3 of the present disclosure, the present disclosure provides a composition comprising either (I) the double-stranded oligonucleotide according to aspect 1, and / or (II) the conjugate according to aspect 2. (I) the double-stranded oligonucleotide according to aspect 1, and / or (II) the conjugate according to aspect 2.

[0014] In aspect 4 of the present disclosure, the present disclosure provides the use of any of (I) the double-stranded oligonucleotide according to aspect 1, and / or (II) the conjugate according to aspect 2, and / or (III) the composition according to aspect 3, in the preparation of a medicament for preventing and / or treating a disease or disorder mediated by the INHBE gene. (I) the double-stranded oligonucleotide according to aspect 1, and / or (II) the conjugate according to aspect 2, and / or (III) the composition according to aspect 3, in the preparation of a medicament for preventing and / or treating a disease or disorder mediated by the INHBE gene.

[0015] In some preferred embodiments of the present disclosure, the disease or disorder includes, but is not limited to, metabolic disorders, type 2 diabetes, obesity, elevated triglyceride levels, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes, non-alcoholic steatohepatitis (NASH), cardiovascular disease, cardiomyopathy, hypertension, and / or the risk of having or developing heart failure.

[0016] In aspect 5 of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any of (I) the double-stranded oligonucleotide according to aspect 1, and / or (II) the conjugate according to aspect 2, and / or (III) the composition according to aspect 3, and a pharmaceutically acceptable adjuvant or excipient. (I) the double-stranded oligonucleotide according to aspect 1, and / or (II) the conjugate according to aspect 2, and / or (III) the composition according to aspect 3, and a pharmaceutically acceptable adjuvant or excipient.

[0017] In aspect 6 of this disclosure, this disclosure is: (I) Double-stranded oligonucleotides as described in Embodiment 1, and / or (II) Conjugates as described in Embodiment 2, and / or (III) The composition described in embodiment 3, and / or (IV) A method for reducing the expression or activity of the INHBE gene is provided, comprising contacting any of the pharmaceutical compositions described in Embodiment 5 with cells.

[0018] In aspect 7 of this disclosure, this disclosure is: (I) Double-stranded oligonucleotides as described in Embodiment 1, and / or (II) Conjugates as described in Embodiment 2, and / or (III) The composition described in embodiment 3, and / or (IV) A method to prevent and / or treat an INHBE gene-mediated disease or condition, comprising administering to a subject in a pharmaceutically acceptable amount any of the pharmaceutical compositions described in Embodiment 5.

[0019] It is obvious to those skilled in the art that nucleotide groups modified using nucleotide monomers having the corresponding modifications can be introduced into double-stranded oligonucleotide conjugates according to the present invention. Methods for preparing nucleotide monomers having the corresponding modifications and methods for introducing modified nucleotide groups into double-stranded oligonucleotide conjugates are also well known to those skilled in the art. All modified nucleotide monomers may be commercially available or prepared by known methods. [Brief explanation of the drawing]

[0020] [Figure 1] This describes the suppressive activity of target genes in Balb / c-HDI mice in vivo after administration of an siRNA conjugate. [Figure 2]This describes the suppressive activity of target genes in C57BL / 6J mice in vivo after administration of siRNA conjugates. [Figure 3] This shows the suppressive activity of target genes in C57BL / 6J mice after administration of different doses of RZM08019. [Figure 4] This is the triglyceride level in BKS-DB mouse serum after repeated administration of RZM08019. [Figure 5] This is the total cholesterol level in the serum of BKS-DB mice after repeated administration of RZM08019. [Figure 6] This refers to the suppressive activity of target genes in Huh7 cells after administration of siRNA conjugates. [Modes for carrying out the invention]

[0021] The following describes the technical concepts in the embodiments of this disclosure clearly and completely, and it is clear that the embodiments described are not all embodiments, but only a selection of embodiments of this disclosure. All other embodiments obtained by a person skilled in the art without any creative work, according to the embodiments of this disclosure, fall within the scope of protection of this disclosure.

[0022] Interpretation of terms Unless otherwise specified, the following definitions used herein should be used. For the purposes of this disclosure, chemical elements and the CAS version of the periodic table correspond to the Handbook of Chemistry and Physics, 75th edition, 1994. General principles of organic chemistry can also be referenced from “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry” by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, all of which are incorporated herein by reference. Alkylene groups may be substituted or unsubstituted.

[0023] As used in this disclosure, “halogen” or “halogeno” means any of the radioactive-stable atoms in the seventh column of the periodic table, for example, fluorine, chlorine, bromine, or iodine. Here, fluorine and chlorine are preferred, and fluorine is more preferred.

[0024] As used in this disclosure, “alkyl group” means a fully saturated (i.e., non-double or triple bonded) linear or branched alkane chain. An alkyl group may have 1 to 6 carbon atoms (wherein it appears, a numerical range such as “1 to 6” means each integer within a given range; for example, “1 to 6 carbon atoms” means that an alkyl group can consist of 1, 2, 3, 4, 5, or 6 carbon atoms, although this definition includes the term “alkyl group” without a specified numerical range). As a mere example, “C1-C4 alkyl group” means that the alkyl chain has 1 to 4 carbon atoms, i.e., the C1-C4 alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, s-butyl, or t-butyl groups. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, and hexyl groups. The alkyl group may be substituted or unsubstituted.

[0025] As used in this disclosure, "alkoxy group" means formula -OR, where R is an alkyl group as described above, for example, a "C1-6 alkoxy group," and includes, but is not limited to, a methoxy group, an ethoxy group, an n-propoxy group, a 1-methylethoxy group (isopropoxy group), an n-butoxy group, an isobutoxy group, an s-butoxy group, and a t-butoxy group.

[0026] As used in this disclosure, "alkylene group" means a linear or branched alkylene chain with the formula -R- that is completely saturated. As a mere example, "C1-10 alkylene group" indicates that the alkyl chain has 1 to 10 carbon atoms, that is, the C1-10 alkylene group is selected from methylene group (-CH2-), ethylene group (=CH2CH3), 1,2-ethylene group (-CH2CH2-), n-propylene group (-CH2CH2CH2-), isopropylene group (-CH2CH(CH3)-), etc.

[0027] As used in this disclosure, the articles “1,” “one (kind),” and “the foregoing” are intended to include “at least one” or “one or more.” Accordingly, as used herein, these articles mean articles of one or more than one (i.e., at least one) object. For example, “one component” means one or more components, i.e., more than one component may be employed or used in the embodiment described above.

[0028] In this disclosure, the terms “equip,” “include,” “have,” “possible,” “contain,” and their variations are generally intended to be open, transitional phrases, terms, or words and do not preclude the possibility of additional actions or structures. The term “composed of…” generally indicates that no other component (or similarly, feature, integer, step, etc.) may exist. Unless otherwise specified in the context, nouns that are not limited in number also include multiple referents.

[0029] In this disclosure, the terms “preferably,” “optionally,” or “optionally” generally mean that the events or circumstances described below may, but will not necessarily, occur, and such descriptions include both cases where such events or circumstances occur and cases where such events or circumstances do not occur.

[0030] The term "includes" as used in this disclosure is an open expression, meaning it includes the content explicitly stated in this disclosure but does not exclude content in any other form.

[0031] As used in this disclosure, “optionally substituted” is used to define a variable, which may be non-substituted.

[0032] As used in this disclosure, “unsubstituted” means that the specified group is unsubstituted.

[0033] As used in this disclosure, “substituted,” “substituted,” and “substituted” are interchangeable, meaning that one or more hydrogen atoms in a given structure are substituted by a specific substituent. Unless otherwise shown, one substituted group can be substituted by one substituent at each substituted position of the group. If one or more positions in a given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may substituted similarly or differently at each substituted position.

[0034] The phrases "each is independently chosen from...", "each is independently chosen from...", and "...is independently chosen from..." as used in this disclosure are interchangeable and should all be interpreted broadly. They may mean that specific choices represented by the same symbols on different bases do not influence each other, or that specific choices represented by the same symbols on the same base do not influence each other.

[0035] In this disclosure, the terms “equip,” “include,” “have,” “possible,” “contain,” and their variations are generally intended to be open, transitional phrases, terms, or words and do not preclude the possibility of additional actions or structures. The term “composed of…” generally indicates that no other component (or similarly, feature, integer, step, etc.) may exist. Unless otherwise specified in the context, nouns that are not limited in number also include multiple referents.

[0036] As used in this disclosure, "small interfering RNA (siRNA)" is a type of double-stranded RNA, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA induces specific degradation of mRNA sequences through known RNA interference (RNAi) processes, thereby suppressing the translation of mRNA into amino acids and its conversion into proteins.

[0037] In this disclosure, a double-stranded oligonucleotide consists of two strands, where the strand linked at the same target sequence is called the antisense strand or guide strand, and the other strand is called the sense strand or passenger strand. The term “antisense strand” means a single strand, such as a double-stranded oligonucleotide, which contains a region that is fully or nearly complementary to the target sequence. The term “sense strand” means a single strand, such as a double-stranded oligonucleotide, which contains a region that is nearly complementary to the region of the antisense strand as defined herein. The term “complementary region” means a region in the antisense strand that is fully or nearly complementary to the target sequence. If the complementary region and the target sequence are not fully complementary, the mismatch may be located in the interior or terminal region of the molecule. As used herein, the term “complementary” means the ability of the first polynucleotide to hybridize with the second polynucleotide under certain conditions (e.g., strict conditions). In this specification, double-stranded oligonucleotides and siRNAs may be used interchangeably.

[0038] In this disclosure, the terms “complementary” and “reverse complementary” are interchangeable and have meanings familiar to those skilled in the art, namely, in a double-stranded nucleic acid molecule, the bases in one strand are paired in a complementary manner with the bases in the other strand.

[0039] In this disclosure, unless otherwise specified, “nearly inversely complementary” or “nearly complementary” means that there are three or fewer base mismatches between the nucleotide sequences of the two segments, “substantially inversely complementary” means that there is one or fewer base mismatches between the nucleotide sequences of the two segments, and “fully inversely complementary” means that there are no base mismatches between the nucleotide sequences of the two segments.

[0040] In this disclosure, a “nucleotide difference” between one nucleotide sequence and another means that the type of base of the nucleotide at the same position in the former has changed compared to the latter. For example, if one nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference is recognized between the two nucleotide sequences at that position. In some embodiments, if the nucleotide at the original position is replaced with a baseless nucleotide or its equivalent, a nucleotide difference may be considered to have occurred at that position.

[0041] In this disclosure, "fluoro-modified nucleotide" or "2'-fluoro-modified nucleotide" means a nucleotide formed by substituting the hydroxyl group at the 2' position of the ribosyl group with fluorine, and "unfluoro-modified nucleotide" means a nucleotide or nucleotide analog formed by substituting the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group. The "methoxy-modified nucleotide" or "2'-O-methoxyethyl-modified nucleotide" means a nucleotide formed by substituting the hydroxyl group at the 2' position of the ribosyl group with a methoxy group or a methoxyethyl group, and "methoxy-modified nucleotide" is also described as a nucleotide modified with a 2'-OMe or 2'-O-methyl group, and "2'-O-methoxyethyl-modified nucleotide," which can be used interchangeably, is also described as 2'-MOE modification, and 2'-deoxynucleotide means that the 2' position of the ribosyl group is hydrogen.

[0042] The terms “RNAi,” “iRNA,” “RNAi agent,” “RNAi reagent,” “RNA interference agent,” and “RNA inhibitor” as used herein are interchangeable, meaning they include RNA molecules or reagents that can mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. It is well known in the art that RNA induces specific degradation of mRNA sequences through a process known as RNA interference (RNAi). In one embodiment, the RNAi agent of this disclosure includes single-stranded or double-stranded RNA that interacts with a target RNA sequence to induce cleavage of the target RNA. Accordingly, in one embodiment, the term “siRNA” as used herein may also be used to refer to the above-mentioned RNAi.

[0043] In some embodiments, “RNAi” as used in the compositions, uses, and methods of the present disclosure is double-stranded RNA, and “RNAi agent” includes said double-stranded RNA, and “RNAi agent” may mean “double-stranded RNAi reagent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA reagent,” “siRNA reagent,” or “dsRNA agent.”

[0044] In this disclosure, the terms “dsRNA” or “siRNA” mean a complex of ribose nucleic acid molecules, comprising two antiparallel and nearly complementary nucleic acid strands having a double-stranded structure and having “sense” and “antisense” orientations with respect to the target RNA.

[0045] Furthermore, as used herein, “RNAi reagent” or “RNAi agent” may include ribonucleotides having chemical modifications and / or ligands, and RNAi reagents may include substantial modifications in multiple nucleotides. The term “modified nucleotide” means a nucleotide having independently modified sugar moieties, modified internucleotide bonds and / or modified nucleic acid bases. Accordingly, the term modified nucleotide includes substitution, addition or removal of, for example, functional groups or atoms to internucleotide bonds, sugars or nucleic acid bases. The modifications applicable to the reagents of this disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, as used for siRNA molecules, can be covered by an “RNAi agent.”

[0046] In this disclosure, the terms “nucleotide overhang” or “suspension” mean at least one unpaired nucleotide overhanging from the double-stranded structure of an iRNA (e.g., dsRNA). For example, a nucleotide overhang exists if the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. One or more overhangs may be present on the sense strand, the antisense strand, or any combination thereof. Alternatively, one or more nucleotides of an overhang may be present on the 5' end, 3' end, or both ends of the antisense or sense strand of dsRNA.

[0047] In this disclosure, the term “ligand” typically means a compound or molecule that can be covalently bound to or otherwise chemically bound to a bioactive substance (e.g., an oligonucleotide). In some embodiments, a ligand can interact directly or indirectly with another compound (e.g., a receptor), the receptor interacting with the ligand may be located on the cell surface or may be an intracellular and / or intercellular receptor, and the interaction between the ligand and the receptor may result in a biochemical reaction or may be merely a physical interaction or binding.

[0048] The term "covalent" refers to a connection between two molecules, meaning that the two molecules are linked via covalent bonds or via non-covalent bonds (e.g., hydrogen bonds or ionic bonds).

[0049] Each nucleotide in the sense strand and antisense strand is independently modified or unmodified. In the context of this invention, unless otherwise specified, "complex" means that two or more chemical moieties, each having a specific function, are covalently linked to one another, and correspondingly, "conjugate" means a compound formed by the covalent bonding of such chemical moieties. Furthermore, "siRNA conjugate" means a compound formed by the covalent bonding of one or more chemical moieties, each having a specific function, to an siRNA. Hereinafter, the siRNA conjugate of this invention may be abbreviated as "conjugate". Depending on the context, "siRNA conjugate" should be understood as a general term for siRNA conjugates, a first-type siRNA conjugate or a second-type siRNA conjugate, or an siRNA sense strand conjugate or an siRNA antisense strand conjugate.

[0050] In this disclosure, the terms “pharmaceutical composition” or “composition” may be used for the treatment of a disease or for in vitro cell culture experiments. When used for the treatment of a disease, the term “pharmaceutical composition” usually means in the form of a unit dose and can be prepared by any method well known in the pharmaceutical field. All methods include a step of conjugating an active ingredient with one or more adjuvants constituting accessory components. Typically, compositions are prepared by uniformly and sufficiently conjugating active siRNA with a liquid adjuvant, a finely ground solid adjuvant, or both.

[0051] In this disclosure, the term “pharmaceutically acceptable” means that a substance or composition must be chemically and / or toxicologically compatible with other components, including the formulation, and / or with the mammal it treats. Preferably, “pharmaceutically acceptable” as used in this disclosure means for use in animals approved by a federal supervisory agency or national government, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias, particularly for use in the human body.

[0052] Any “pharmaceutically acceptable carrier or auxiliary agent” used in this disclosure may include any solvent, solid excipient, diluent or other liquid excipient, etc., and is suitable for a specific target dosage form. In addition to the extent to which any common auxiliary agent is incompatible with the siRNA of this disclosure, any adverse biological effects produced or interactions produced in an adverse manner by any other component of the pharmaceutically acceptable composition are also considered in this disclosure.

[0053] As used in this disclosure, “treatment,” “alleviation,” or “improvement” are interchangeable herein. These terms mean a method of obtaining a beneficial or desired outcome, including but not limited to therapeutic benefit. “Therapeutic benefit” means eradicating or improving the potential disorder being treated. Also, while a subject may suffer from a potential disorder, therapeutic benefit is obtained by observing improvement in the subject by eradicating or improving one or more physiological symptoms associated with the potential disorder.

[0054] The terms “prevention” and “prevention” as used in the disclosure are interchangeable, and the methods for obtaining beneficial or desired results include, but are not limited to, preventive benefits. To obtain “preventive benefits,” a conjugate, RNAi reagent, or composition may be administered to a subject at risk of developing a particular disease or to a subject reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease may not yet have been made.

[0055] In this disclosure, the term “administration” typically means introducing the pharmaceutical formulations of this disclosure into the body of a subject via any introduction or delivery route. Any method known to those skilled in the art can be employed to bring cells, organs or tissues into contact with the drug. Such administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. A daily dose may be divided into one, two or more appropriate forms of dose for administration at one, two or more time intervals within a given time period.

[0056] In this disclosure, the term “contact” generally means contact between two or more different types of substances in any order, in any manner and for any duration. Contact can occur in vivo, ex vivo, or in vitro. In some embodiments, it may mean direct contact between the RNAi agent or composition of this disclosure and cells or tissues. In other embodiments, the term may mean indirect contact between the RNAi agent or composition of this disclosure and cells or tissues.

[0057] In this disclosure, the term “Subject” typically means a human or non-human animal (including mammals) that needs to be diagnosed, prognosed, improved, prevented and / or treated for a disease, such as humans, non-human primates (monkeys, gibbons, gorillas, chimpanzees, orangutans, rhesus monkeys), domesticated animals (dogs and cats), livestock animals (horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, neonates, infants, adolescents, and adult subjects. Subjects include animal disease models.

[0058] In this disclosure, the term “modulate gene expression” means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is regulated to a higher or lower degree than that observed in the absence of the regulator. For example, the term “modulate” may mean “suppress,” but the use of the word “modulate” is not limited to this definition.

[0059] In addition to any conventional adjuvants, any adverse biological effects produced or adverse interactions produced with any other components of a pharmaceutically acceptable composition, to the extent incompatible with the siRNA of this disclosure, are also considered within the scope of this disclosure.

[0060] Double-stranded oligonucleotides targeting the INHBE gene In one aspect of this disclosure, the disclosure provides a double-stranded oligonucleotide that targets the INHBE gene and can suppress INHBE gene expression in mammals, including humans, monkeys, rats, or mice. The double-stranded oligonucleotide can suppress intracellular INHBE gene expression in vitro and can also suppress INHBE gene expression in vivo.

[0061] Specifically, the present disclosure provides a double-stranded oligonucleotide targeting the INHBE gene, comprising a sense strand and an antisense strand, wherein the antisense strand and the sense strand are complementary or nearly complementary, with nearly complementary meaning that there are three or fewer nucleotide mismatches in the double-stranded regions of the sense strand and the antisense strand.

[0062] Here, the sense strand contains a nucleotide sequence that is the same as or nearly the same as at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence, where "nearly the same" means that there is a difference of three or fewer nucleotides between the sense strand and the at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence.

[0063] In preferred embodiments of the present disclosure, the sense strand comprises a nucleotide sequence in which the difference between at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence is 2 or less, preferably with a difference of 1 or less.

[0064] In preferred embodiments of the present disclosure, the antisense strand is complementary or nearly complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the nucleotide sequence of SEQ ID NO:309, wherein nearly complementary means that there are three or fewer nucleotide mismatches in the complementary region.

[0065] In preferred embodiments of the present disclosure, the double-stranded oligonucleotide comprises one sense strand and one antisense strand, the antisense strand comprising a nucleotide sequence in which at least 15 consecutive nucleotides are in any of the sequences shown in Table 1 as SEQ ID NO:155 to SEQ ID NO:308, or a nucleotide sequence in which the difference between the at least 15 consecutive nucleotides is 3 or less.

[0066] The sense strand includes a nucleotide sequence that forms a double-stranded region at least partially inversely complementary or nearly complementary with the antisense strand, where nearly complementary means that there are three or fewer nucleotide mismatches in the double-stranded regions of the sense strand and the antisense strand.

[0067] In some preferred embodiments of the present disclosure, the antisense strand comprises a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides in any of the sequences shown in SEQ ID NO:155 to SEQ ID NO:308 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is 3 or less.

[0068] In some preferred embodiments of the present disclosure, the antisense strand comprises a nucleotide sequence in which at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides in any of the sequences shown in SEQ ID NO:155 to SEQ ID NO:308 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is two or less.

[0069] In some preferred embodiments of the present disclosure, the antisense strand comprises a nucleotide sequence in which at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides in any of the sequences shown in SEQ ID NO:155 to SEQ ID NO:308 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is one or less.

[0070] In some preferred embodiments of the present disclosure, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, and at least 21 consecutive nucleotides in any of the nucleotide sequences shown in SEQ ID NO. 155 to SEQ ID NO. 308 in Table 1.

[0071] In some specific embodiments of this disclosure, the antisense strand is selected from or includes any of the nucleotide sequences shown in SEQ ID NO. 155 to SEQ ID NO. 308 in Table 1.

[0072] In a preferred embodiment of the present disclosure, in the direction of 5'→3', positions 2 to 19 of the antisense strand include at least 15 nucleotides from positions 2 to 19 of any nucleotide sequence shown in SEQ ID NO. 155 to SEQ ID NO. 308 in Table 1, or a nucleotide sequence in which the difference from said at least 15 nucleotides is 3 or less.

[0073] In some preferred embodiments of the present disclosure, the sense strand includes a nucleotide sequence in which at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides in any of the sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is 3 or less.

[0074] In some preferred embodiments of the present disclosure, the sense strand includes a nucleotide sequence in which at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides in any of the sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is two or less.

[0075] In some preferred embodiments of the present disclosure, the sense strand includes a nucleotide sequence in which at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides in any of the sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is one or less.

[0076] In some preferred embodiments of the present disclosure, the sense strand comprises at least 15, at least 16, at least 17, at least 18, and at least 19 consecutive nucleotides in any of the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1.

[0077] In some specific embodiments of this disclosure, the sense strand is selected from or includes any of the nucleotide sequences shown in Table 1 as SEQ ID NO.1 to SEQ ID NO.154.

[0078] In some preferred embodiments of the present disclosure, the double-stranded oligonucleotides include one or more species within the double-stranded group indicated by the numbers in Table 1, namely RN008015, RN008125, RN008128, RN008142, RN008145, and RN008148.

[0079] In some preferred embodiments of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0080] Here, the structural formula of a nucleotide is

[0081] [ka] Hereinafter, Base represents a nucleotide base, and each nucleotide base on a nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A, or guanine G.

[0082] In some preferred embodiments of the present disclosure, almost all nucleotides of the sense strand or the antisense strand are selected from modified nucleotides. Here, “almost all nucleotides of the sense strand are selected from modified nucleotides” means the majority of nucleotides in the sense strand, but not all of them, and may include 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0083] In some specific embodiments of the present disclosure, all nucleotides of the sense strand or the antisense strand are selected from modified nucleotides.

[0084] In some preferred embodiments of this disclosure, the modified nucleotides are each independently 2'-halogen, 2'-deoxy, and 2'-O-(CH2) n -R1 modified nucleotides or nucleotide analogs are selected, and the nucleotide analogs are one or more selected from peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), ethylene glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA), and unlocked nucleic acid (UNA).

[0085] n is chosen from 0, 1, or 2, and R1 is a C which can be arbitrarily substituted. 1~6 Alkyl group, optionally substituted C 1~6 Alkoxy group or -Si(R 1a)Selected from 3, each R 1a is an independently and optionally substituted C 1~6 alkyl group or an optionally substituted C 1~6 alkoxy group.

[0086] In the present disclosure, a nucleotide modified with 2'-halogen has a hydroxy group at the 2'-position substituted by a halogen atom. Exemplarily, a nucleotide modified with 2'-fluoro has a structural formula of

[0087]

Chemical formula

[0088] In the present disclosure, a nucleotide modified with 2'-deoxy has a hydroxy group at the 2'-position of the nucleotide substituted by a hydrogen atom. Its structural formula is

[0089]

Chemical formula

[0090] In the present disclosure, a nucleotide modified with 2'-O-(CH2) n -R1 has a hydrogen atom at the hydroxy group at the 2'-position of the nucleotide substituted by -(CH2) n -R1, where the structural formula of the nucleotide modified with 2'-O-(CH2) n -R1 is

[0091]

Chemical formula

[0092] [ka] The structural formula of TIPS is

[0093] [ka] Therefore, the structural formula of TOM is

[0094] [ka] That is the case.

[0095] In some preferred embodiments of this disclosure, 2'-O-(CH2) n -R1 is selected from 2'-O-CH3, 2'-O-CH2-O-CH3, 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3, or 2'-O-CH2-CH2-O-CH3.

[0096] In some preferred embodiments of the present disclosure, the sense strand or the antisense strand includes a 3' overhang having at least one nucleotide.

[0097] In some preferred embodiments of the present disclosure, the antisense strand includes a 3' overhang having at least one nucleotide.

[0098] In some preferred embodiments of the present disclosure, the sense strand or the antisense strand includes a 3' overhang having at least two nucleotides.

[0099] In some preferred embodiments of the present disclosure, the antisense strand includes a 3' overhang having at least two nucleotides.

[0100] In some specific embodiments of the present disclosure, the antisense strand includes a 3' overhang having two nucleotides.

[0101] In some preferred embodiments of the present disclosure, the sense chain and / or the antisense chain independently comprises one or more thiophosphate bonds.

[0102] In some specific embodiments of the present disclosure, the sense strand includes two consecutive thiophosphate bonds between the terminal nucleotides at the 5' end.

[0103] In some specific embodiments of the present disclosure, the antisense strand comprises two consecutive thiophosphate bonds between the 3' and 5' terminal nucleotides, respectively.

[0104] In some preferred embodiments of the present disclosure, all nucleotides of the sense strand and all nucleotides of the antisense strand are selected from modified nucleotides, where the double-stranded region formed by the sense strand and the antisense strand is as shown in formula (I) below. SS:5'-(N)a' -(X)p' -(N)b' -(X)q' -(N)c' -(X)r' -(N)d' -3' AS:3' -(N)a-(X)p-(N)b-(X)q-(N)c-5'(I), However, SS represents the sense chain, and AS represents the antisense chain. Each N is independently selected from nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, or nucleotides modified with 2'-deoxy. Each X is independently selected from 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3, and 2'-O-CH2-CH2-O-CH3. The above a, a', p, p', b, b', q, q', c, c', r', and d' each independently represent the number of nucleotides, where a' is selected from an integer between 3 and 8, p' is selected from an integer between 0 and 3, b' is selected from an integer between 4 and 13, q' is selected from an integer between 0 and 4, c' is selected from an integer between 3 and 9, r' is selected from an integer between 0 and 3, d' is selected from an integer between 0 and 9, a is selected from an integer between 4 and 7, p is selected from an integer between 0 and 1, b is selected from an integer between 4 and 8, q is selected from an integer between 0 and 4, and c is selected from an integer between 6 and 10, and p', q', r', p, and q cannot be 0 at the same time, and 0 ≤ q' + r' ≤ 4.

[0105] In some preferred embodiments of the present disclosure, a' is selected from an integer between 3 and 8, p' is selected from 0 or 1, b' is selected from an integer between 4 and 13, q' is selected from 0 or 1, c' is selected from an integer between 3 and 9, r' is selected from 0 or 1, d' is selected from an integer between 1 and 8, a is selected from an integer between 4 and 7, p is selected from 1, b is selected from an integer between 4 and 8, q is selected from 0 or 1, and c is selected from an integer between 6 and 10.

[0106] In some preferred embodiments of this disclosure, each N is independently selected from 2'-O-methyl-modified nucleotides and 2'-fluoro-modified nucleotides.

[0107] In some preferred embodiments of the present disclosure, each X is independently selected from nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-MOE (methoxyethyl), nucleotides modified with 2'-O-TBDMS, nucleotides modified with 2'-O-TIPS, nucleotides modified with 2'-O-TOM, nucleotides modified with 2'-O-CH2-O-CH2-CH3, and nucleotides modified with 2'-O-CH2-O-CH2-CF3.

[0108] Preferably, each X is independently selected from nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-MOE, nucleotides modified with 2'-O-CH2-O-CH2-CH3, and nucleotides modified with 2'-O-CH2-O-CH2-CF3.

[0109] In some preferred embodiments of the present disclosure, the double-stranded region comprises at least one nucleotide modified with 2'-O-methoxyethyl or nucleotide modified with 2'-O-ethoxymethyl.

[0110] In some preferred embodiments of the present disclosure, the double-stranded region comprises at least one nucleotide modified with 2'-O-methoxyethyl.

[0111] In some specific embodiments of the present disclosure, the double-stranded region comprises a nucleotide modified with one 2'-O-methoxyethyl molecule.

[0112] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0113] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0114] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0115] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0116] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0117] In some preferred embodiments of the present disclosure, the antisense chain comprises at least one nucleotide modified with 2'-O-methoxyethyl or 2'-O-ethoxymethyl.

[0118] In some preferred embodiments of the present disclosure, the antisense chain comprises at least one nucleotide modified with 2'-O-methoxyethyl.

[0119] In some specific embodiments of the present disclosure, the antisense chain includes a nucleotide modified with one 2'-O-methoxyethyl molecule.

[0120] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from a nucleotide modified with 2'-O-methoxyethyl, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl.

[0121] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from a nucleotide modified with 2'-O-methoxyethyl, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl.

[0122] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from a nucleotide modified with 2'-O-methoxyethyl, any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl.

[0123] In some specific embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from nucleotides modified with 2'-O-methoxyethyl, the nucleotides at positions 2, 6, 9, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl.

[0124] In some specific embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from nucleotides modified with 2'-O-methoxyethyl, the nucleotides at positions 2, 6, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl.

[0125] In some preferred embodiments of the present disclosure, in a direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7-10 of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0126] In some specific embodiments of the present disclosure, in a direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl.

[0127] In some preferred embodiments of the present disclosure, the antisense chain comprises a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides in any modified antisense chain nucleotide sequence shown in Table 2, or a nucleotide sequence in which the difference between such consecutive nucleotides is 3 or less.

[0128] In some preferred embodiments of the present disclosure, the antisense strand comprises a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides in any modified antisense strand nucleotide sequence shown in Table 2, or a nucleotide sequence in which the difference between such consecutive nucleotides is two or less.

[0129] In some preferred embodiments of the present disclosure, the antisense chain comprises a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides in any modified antisense chain nucleotide sequence shown in Table 2, or a nucleotide sequence in which the difference between such consecutive nucleotides is one or less.

[0130] In some preferred embodiments of the present disclosure, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, and at least 21 consecutive nucleotides in the nucleotide sequence of any modified antisense strand shown in Table 2.

[0131] In some specific embodiments of this disclosure, the antisense strand is selected from or includes any modified antisense strand nucleotide sequences shown in Table 2.

[0132] In some preferred embodiments of the present disclosure, the sense strand comprises a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides in any modified sense strand nucleotide sequence shown in Table 2, or a nucleotide sequence in which the difference between such consecutive nucleotides is one, two, or three nucleotides.

[0133] In some specific embodiments of the present disclosure, the sense strand of the double-stranded oligonucleotide is selected from or includes any modified sense strand nucleotide sequences shown in Table 2.

[0134] In some specific embodiments of the present disclosure, the sense strand of the double-stranded oligonucleotide is selected from or includes any modified sense strand nucleotide sequences shown in Table 2, and the antisense strand is selected from or includes any modified antisense strand nucleotide sequences shown in Table 2.

[0135] In preferred embodiments of the present disclosure, the double-stranded oligonucleotide is selected from or comprises one or more of the double-stranded groups modified by the numbers in Table 2, namely RX008157, RX008174, RX008177, RX008180, RX008182, and RX008185.

[0136] In some specific embodiments of this disclosure, the double-stranded oligonucleotide is selected from siRNA.

[0137] Double-stranded oligonucleotide conjugates In aspect 2 of the present disclosure, the present disclosure provides a double-stranded oligonucleotide conjugate comprising a double-stranded oligonucleotide as described in aspect 1 and a ligand capable of binding to one or more cell receptors.

[0138] In some preferred embodiments of the present disclosure, the ligand is compounded with the sense chain and / or the antisense chain.

[0139] In some preferred embodiments of the present disclosure, the ligand is compounded to the 3' and / or 5' ends of the sense strand.

[0140] In some specific embodiments of this disclosure, the ligand is compounded at the 3' end of the sense chain.

[0141] In some specific embodiments of this disclosure, the cell receptor is selected from asialoclycoprotein receptors.

[0142] In some specific embodiments of the present disclosure, the ligand is selected from a galactose cluster. The galactose cluster comprises molecules having 2 to 4 terminal galactose derivatives. The galactose derivatives used in the present disclosure include galactose derivatives having an affinity for galactose and / or asialoclycoprotein receptors equal to or greater than that of galactose.

[0143] In some specific embodiments of the present disclosure, the galactose cluster comprises molecules having 2 to 4 terminal N-acetylgalactosamines (GalNAc).

[0144] In some preferred embodiments of the present disclosure, the ligand is selected from the structure represented by formula (101) or its isomers or pharmaceutically acceptable salts thereof.

[0145] [ka] however, * This represents a composite site between the ligand and the sense chain or the antisense chain, m is selected from 1, 2, 3, or 4. Each Z is independently selected from either a hydroxyl group or a mercapto group. Each p is independently chosen from 1, 2, or 3. Each q is independently selected from 1, 2, or 3. Each R is independently selected from H, an optionally substituted C1-C6 alkyl group, or an optionally substituted C1-C6 alkoxy group. Each L is an independently and optionally substituted C2-C20 alkylene group or

[0146] [ka] Selected from, R La and R Lb k is independently and arbitrarily selected from C1-C10 alkylene groups, and k is selected from 1, 2, 3, 4 or 5. Each Y is independently selected from O, S, or NH.

[0147] In some preferred embodiments of this disclosure, m is selected from 1, 2, or 3.

[0148] In some specific embodiments of this disclosure, m is selected from 3.

[0149] In some specific embodiments of this disclosure, Z is selected from a hydroxyl group.

[0150] In some specific embodiments of this disclosure, p is selected from 1.

[0151] In some specific embodiments of this disclosure, q is selected from 1.

[0152] In some specific embodiments of this disclosure, R is selected from H.

[0153] In some preferred embodiments of this disclosure, each L is independently a C1-C10 alkylene group or

[0154] [ka] Selected from, however, R La and R Lb k is independently selected from C1-C5 alkylene groups, and k is 1, 2, or 3.

[0155] In some specific embodiments of this disclosure, k is selected from 1, In some preferred embodiments of this disclosure, each L is independently

[0156] [ka] They are selected from among them.

[0157] In some specific embodiments of this disclosure, Y is selected from O.

[0158] In some preferred embodiments of this disclosure, the ligand is

[0159] [ka] This includes at least one of the following structures, its isomers, or pharmaceutically acceptable salts thereof.

[0160] In some specific embodiments of this disclosure, the ligand is

[0161] [ka] The structure is selected from its isomers or pharmaceutically acceptable salts.

[0162] In some preferred embodiments of the present disclosure, the double-stranded oligonucleotide conjugate is one or more selected from the conjugates indicated by the numbers RZ08040, RZ08041, RZ08042, RZ08043, RZ08044, and RZ08045 in Table 4.

[0163] In some specific embodiments of this disclosure, the ligand is selected from the structure represented by formula (201).

[0164] [ka] however, * This represents a composite site between the ligand and the sense chain or the antisense chain.

[0165] composition In aspect 3 of this disclosure, this disclosure is: (I) Double-stranded oligonucleotides as described in Embodiment 1, and / or (II) Provides a composition comprising any of the conjugates described in Embodiment 2.

[0166] Use for disease treatment In aspect 4 of this disclosure, this disclosure is: (I) Double-stranded oligonucleotides as described in Embodiment 1, and / or (II) Conjugates as described in Embodiment 2, and / or (III) The composition described in Embodiment 3 is provided for use in the preparation of a drug for preventing and / or treating a disease or condition mediated by any of the INHBE genes.

[0167] In some preferred embodiments of this disclosure, the disease or condition includes, but is not limited to, metabolic disorders, type 2 diabetes, obesity, elevated triglyceride levels, malnutrition, hepatitis, fatty liver disease, hypercholesterolemia, elevated liver enzymes, non-alcoholic steatohepatitis (NASH), cardiovascular disease, cardiomyopathy, hypertension and / or heart failure, or the risk of developing such a condition.

[0168] Pharmaceutical composition In aspect 5 of this disclosure, this disclosure is: (I) Double-stranded oligonucleotides as described in Embodiment 1, and / or (II) Conjugates as described in Embodiment 2, and / or (III) Provided are pharmaceutical compositions comprising any of the compositions described in Embodiment 3 and a pharmaceutically acceptable auxiliary or adjuvant.

[0169] Methods for reducing INHBE gene expression or activity in vitro In aspect 6 of this disclosure, this disclosure is: (I) Double-stranded oligonucleotides as described in Embodiment 1, and / or (II) Conjugates as described in Embodiment 2, and / or (III) The composition described in embodiment 3, and / or (IV) A method for reducing the expression or activity of the INHBE gene is provided, comprising contacting any of the pharmaceutical compositions described in Embodiment 5 with cells.

[0170] Treatment methods for diseases In aspect 7 of this disclosure, this disclosure is: (I) Double-stranded oligonucleotides as described in Embodiment 1, and / or (II) Conjugates as described in Embodiment 2, and / or (III) The composition described in embodiment 3, and / or (IV) A method to prevent and / or treat an INHBE gene-mediated disease or condition, comprising administering to a subject in a pharmaceutically acceptable amount any of the pharmaceutical compositions described in Embodiment 5.

[0171] The mRNA sequence of the coding INHBE gene according to the present invention (SEQ ID NO:309, NM_031479.5) is as follows:

[0172] [Table 1]

[0173] [Table 2]

[0174] Unless otherwise specified, the proportions of reagents used in each example of this disclosure are calculated as volume ratios (v / v).

[0175] Unless otherwise specified, the reagents used in each example of this disclosure were purchased from Beijing Gonghe Technology Co., Ltd., and the information on the main reagents is as follows.

[0176] [Table 3]

[0177] Ligand preparation Preparation Example 1: Preparation of Compound CR01008

[0178] (1.1) Synthesis of compound CR01008

[0179] [ka]

[0180] The synthesis route for compound CR01008 was as follows:

[0181] [ka]

[0182] (1.1.1) Synthesis of Compound 2

[0183] [ka] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboaldehyde, 10.0 g, 1.0 eq) and an aqueous formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol. 13 ml of a 45.3% by mass aqueous KOH solution was added dropwise. After the addition was complete, the mixture was stirred at 25°C for 30 minutes, then the temperature was raised to 60°C and refluxed for 2 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, then evaporated under reduced pressure to obtain a white solid crude product. A small amount of water was added to the crude product to form a slurry, which was filtered to obtain a white solid compound 2 (9 g, yield 78.9%). MS-ESI(m / z) = 260[M+H] + .

[0184] (1.1.2) Synthesis of Compound 3

[0185] [ka] Compound 2 (9 g, 1 eq) prepared according to step (1.1.1) was dissolved in 70 ml of 1,4-dioxane, and a 1,4-dioxane solution of hydrogen chloride (45 ml, 4 M) was added. The mixture was stirred at 25°C for 1 hour. After the reaction was complete, the reaction mixture was evaporated under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0186] (1.1.3) Synthesis of Compound 5

[0187] [ka] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq), prepared according to step (1.1.2), were dissolved in 15 ml of DMF, HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under an N2 atmosphere for 3 hours. After the reaction was complete, the reaction mixture was evaporated under reduced pressure and reversed-phase purification (22 vol% aqueous acetonitrile solution) was performed to obtain compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589 [M + H] + .

[0188] (1.1.4) Synthesis of Compound 6

[0189] [ka] Compound 5 (1.54 g, 1.0 eq), prepared according to step (1.1.3), was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C in an ice bath, and DMTrCl (4,4'-dimethoxytriphenylmethyl chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was carried out at 25°C for 3 hours, and 15 ml of methanol was added to the reaction mixture to quench the reaction. After the reaction was complete, the reaction mixture was evaporated under reduced pressure and reversed-phase purification (with a 60 vol% aqueous solution of acetonitrile) was performed to obtain compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (m / z) = 891 [M + H] + .

[0190] (1.1.5) Synthesis of compound CR01008

[0191] [ka] Compound 6 (1.08 g, 1.0 eq), prepared according to step (1.1.4), was dissolved in 20 ml of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The mixture was purged three times with nitrogen gas and stirred at 25°C for 2 hours. After the reaction was complete, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was extracted three times (3 × 20 ml) with 20 ml of dichloromethane. The organic phases were combined, the organic phase was evaporated under reduced pressure, and after reverse-phase purification (72 vol% acetonitrile aqueous solution), the mixture was vacuum-dried for 12 hours to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) = 1091 [M + Na] + .

[0192] 1H NMR(400MHz,DMSO-d6)δ 1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37-1.17(m,5H),1.60-1.40(m,6H),1.68-1.62(m,1H),1.80(s,3H),1.80(s,3H),1.92 (s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58-3.39(m,3H) ,3.69-3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H),5.24 (d,J=3.4Hz,1H),5.78(s,1H),6.93-6.87(m,4H),7.35-7.21(m,7H),7.44-7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0193] Preparation Example 2: Preparation of Compound CR01008Z

[0194] [ka] Compound CR01008Z was obtained by linking compound 6, which is used to synthesize compound CR01008, to a solid support CPG.

[0195] The synthesis route for compound CR01008Z was as follows:

[0196] [ka]

[0197] (1.2.1) Synthesis of Compound 9

[0198] [ka] Compound 6 (500 mg), prepared according to step (1.1.4), was dissolved in 10 ml of dichloromethane. Compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added, the mixture was purged three times with nitrogen gas, and the reaction was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 10¹³ [M + Na] + .

[0199] (1.2.2) Synthesis of compound CR01008Z

[0200] [ka] Compound 9 (50 mg, amino CPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg)), prepared according to step (1.2.1), was placed in a 20 ml sample bottle and shaken for 16 hours. After the reaction was complete, the reaction mixture was filtered to obtain the cake, which was washed once (1 × 10 ml) with 10 ml of acetonitrile and then vacuum-dried. The dried cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were placed in a 20 ml sample bottle and shaken for 6 hours. After the reaction was complete, the reaction mixture was filtered to obtain the cake, which was washed once (1 × 10 ml) with 10 ml of acetonitrile and then vacuum-dried to obtain compound CR01008Z (1.03 g, supported weight 20-30 μmol / g).

[0201] Here, Cap1 and Cap2 are capping reagents, where Cap1 is a 20 vol% N-methylimidazole pyridine / acetonitrile mixture with a volume ratio of pyridine to acetonitrile of 3:5, and Cap2 is a 20 vol% acetic anhydride acetonitrile solution.

[0202] Preparation Example 3: Preparation of Compound CR01013 The synthesis route for compound CR01013 was as follows:

[0203] [ka]

[0204] (1.3.1) Synthesis of Compound 2 Compound 1 (trans-4-(Boc-amino)cyclohexylcarboaldehyde, 4.9 g) was dissolved in 17 ml of methanol, and aqueous formaldehyde solution (4.21 g, concentration 37% by mass) and aqueous sodium hydroxide solution (6.5 ml, concentration 45.3% by mass) were added dropwise. After the addition of these solutions was complete, the temperature was raised to 60°C and the mixture was stirred at 60°C for 2 hours. After the reaction was complete, the reaction solution was cooled to 25°C, and then evaporated under reduced pressure to obtain a white solid crude product. A small amount of water was added to the crude product to form a slurry, which was filtered and dried to obtain a white solid compound 2 (4.8 g, yield 85.9%). ESI-MS (m / z) = 260.2 [M+H]+.

[0205] (1.3.2) Synthesis of Compound 3 Compound 2 (4.8 g), prepared according to step (1.3.1), was dissolved in 25 ml of 1,4-dioxane, and a 1,4-dioxane hydrochloride solution (25 ml, 4 M) was added. The mixture was stirred at 25°C for 2 hours. After the reaction was complete, the reaction solution was evaporated under reduced pressure to obtain compound 3 (3.6 g, 99.4% yield) as a white solid.

[0206] (1.3.3) Synthesis of Compound 11 Compound 3 (3.6g), prepared according to step (1.3.2), was dissolved in 36 ml of DMF. TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g) were added, and the mixture was stirred at 25°C for 16 hours. After the reaction was complete, the reaction solution was placed in 200 ml of saturated sodium bicarbonate aqueous solution, extracted three times (3 × 100 ml) with 100 ml of ethyl acetate, and the organic phases were combined. The organic phase was washed once (1 × 50 ml) with 50 ml of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the organic phase was evaporated under reduced pressure. Normal-phase purification was performed by column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to obtain compound 11 (2.3 g, yield 33.0%) as a white solid. ESI MS (m / z) = 379.5[M+H]+.

[0207] (1.3.4) Synthesis of Compound 12 Compound 11 (2.3 g) prepared according to step (1.3.3) was dissolved in 23 ml of methanol, wet palladium carbon (230 mg, supported weight 10% by mass) was added, and the system was purged three times with hydrogen gas. The reaction was carried out with stirring at 25°C under a hydrogen gas atmosphere (15 psi) for 16 hours. After the reaction was complete, the reaction mixture was filtered to obtain the filtrate, and the filtrate was evaporated and dried under reduced pressure to obtain a yellow, oily compound 12 (1.48 g, yield 99.8%).

[0208] (1.3.5) Synthesis of Compound 13 Compound 12 (1.48 g), prepared according to step (1.3.4), was dissolved in 15 ml of DMF. Triethylamine (TEA, 1.22 g), compound 4 (1.35 g), and HBTU (3.45 g) were added, and the mixture was stirred at 25°C for 16 hours. After the reaction was complete, the reaction solution was placed in 150 ml of saturated sodium bicarbonate aqueous solution, extracted three times (3 × 50 ml) with 50 ml of ethyl acetate, and the organic phases were combined. The organic phase was washed once (1 × 30 ml) with 30 ml of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the organic phase was evaporated under reduced pressure. Reverse-phase purification was performed by column chromatography (C18 column, eluent: water / acetonitrile = 5 / 1, v / v) to obtain compound 13 (1.3 g, yield 31.8%) as a white solid. ESI-MS (m / z): 674.3 [M + H]+.

[0209] (1.3.6) Synthesis of Compound 14 Compound 13 (1.1 g), prepared according to step (1.3.5), was dissolved in 11 ml of pyridine. The reaction system was cooled to 0°C in an ice bath, and DMTrCl (813 mg) was added in several portions at 0°C. The reaction system was stirred at 0°C for 1 hour. After the reaction was complete, methanol was added to the reaction mixture to quench it, the solvent was evaporated, and the mixture was purified by reverse-phase column chromatography (eluent: water / acetonitrile = 1 / 4, v / v) to obtain compound 14 (800 mg, yield 50.3%) as a white solid. ESI-MS (m / z): 976.5[M+H]+.

[0210] (1.3.7) Synthesis of compound CR01013 At 25°C, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM), and 4,5-dicyanoimidazole (DCl, 53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 255.4 mg) were added. The reaction system was purged three times with nitrogen gas and stirred at 25°C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was washed twice (2 × 5 ml) with 5 ml of saturated sodium bicarbonate aqueous solution, then once (1 × 30 ml) with 30 ml of saturated sodium chloride aqueous solution to separate the organic phase. The mixture was dried over anhydrous sodium sulfate, and the organic phase solvent was evaporated under reduced pressure. Normal-phase purification was performed by column chromatography (eluent: dichloromethane / methanol = 20 / 1, v / v) to obtain compound CR01013 (532 mg, yield 80.4%) as a white solid. ESI-MS (m / z): 1176.7[M+H]+.

[0211] 1 H NMR(400MHz,DMSO-d6)δ 0.95-1.05(d,J=6.7Hz,5H),1.06-1.15(q,J=7.6Hz,8H),1.15-1.21(t,J=7.2Hz,14H),1.72-1.80(s,3H),1.84- 1.92(s,3H),1.94-2.07(d,J=16.0Hz,7H),2.07-2.14(s,3H),2.64-2.72(q,J=5.8Hz,2H),2.74-2.89(d,J=8.5Hz ,2H),3.35-3.56(m,4H),3.57-3.70(m,4H),3.71-3.77(s,6H),3.81-3.93(m,1H),3.96-4.09(d,J=6.4Hz,3H),6 .82-6.97(d,J=8.7Hz,4H),7.17-7.27(t,J=8.7Hz,5H),7.27-7.34(t,J=7.6Hz,2H),7.34-7.43(d,J=7.5Hz,2H).

[0212] Preparation Example 4: Synthesis of Compound CR01013Z The synthesis route for compound CR01013Z was as follows:

[0213] [ka]

[0214] (1.4.1) Synthesis of Compound 15 At 25 °C, Compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane, and triethylamine (25.9 mg, 0.25 mmol), DMAP (1.25 mg, 0.01 mmol), and Compound 8 (succinic anhydride, 15.4 mg, 0.15 mmol) were added. The reaction system was stirred at 25 °C for 16 hours. After completion of the reaction, the solvent in the reaction solution was evaporated off, and reverse-phase purification was performed by column chromatography (C18 column, eluent: water / acetonitrile = 2 / 1, v / v) to obtain yellow oily Compound 15 (110 mg, 0.10 mmol, yield 100%). ESI-MS (m / z) = 1099.3 [M+Na]+.

[0215] (1.4.2) Synthesis of Compound CR01013Z Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 ml of acetonitrile, and HBTU (24.2 mg, 0.06 mmol), DIEA (11.0 mg, 0.08 mmol), and amino-CPG (1.06 g, loading amount 80 μmol / g) were added. The reaction system was stirred and reacted at 25 °C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a cake, which was washed twice with 50 ml of dichloromethane (2 × 50 ml), three times with 50 ml of acetonitrile (3 × 50 ml), and once with 50 ml of ethyl acetate (1 × 50 ml), and then dried in vacuo. Cap1 (4.8 ml), Cap2 (0.54 ml), and DMAP (2.59 mg) were added to the dried cake, and the reaction system was stirred and reacted at 25 °C for 5 hours. After completion of the reaction, the reaction solution was filtered to obtain a cake, which was washed three times with 50 ml of acetonitrile (3 × 50 ml) and dried in vacuo to obtain Compound CR01013Z (900 mg, loading amount 20 - 30 μmol / g).

[0216] Here, Cap1 and Cap2 are capping reagents, where Cap1 is a 20 vol% N-methylimidazole pyridine / acetonitrile mixture with a volume ratio of pyridine to acetonitrile of 3:5, and Cap2 is a 20 vol% acetic anhydride acetonitrile solution.

[0217] Compound L96-PS The structural formula of compound L96-PS was as follows:

[0218] [ka]

[0219] However, PS stands for polystyrene resin solid support.

[0220] Preparation of double-stranded oligonucleotides (siRNAs) Preparation Example 5 (1.5.1) Synthesis of Sense Chain SS Using a solid-phase synthesis method for phosphoramidite nucleic acids, nucleotide monomers were linked one by one in a circular manner following the nucleotide sequence, from 3' to 5'. Each linkage of nucleotide monomers involved four reaction steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthesis conditions were determined as follows:

[0221] Nucleotide monomers were prepared in a 0.1 M solution of nucleotide monomers in acetonitrile.

[0222] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were a temperature of 25°C, a reaction time of 70 seconds, a deprotection reagent of dichloroacetic acid in dichloromethane (3 vol%), and a molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group in the solid support of 5:1.

[0223] The coupling reaction conditions were the same for each step. The coupling reaction conditions were: a temperature of 25°C, a molar ratio of nucleic acid sequence linked to the solid support to nucleotide monomers of 1:10, a molar ratio of nucleic acid sequence linked to the solid support to coupling reagent of 1:65, a reaction time of 600 seconds, the coupling reagent being a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile, and the thio reagent being a 0.2 M solution of xanthogen hydride in acetonitrile / pyridine (with a volume ratio of acetonitrile to pyridine of 1:1).

[0224] The conditions for the capping reaction in each step were the same. The capping reaction conditions were: a temperature of 25°C, a reaction time of 2 minutes, a capping reagent solution consisting of a mixed solution of Cap1 and Cap2 in a molar ratio of 1:1, Cap1 being a 20 vol% N-methylimidazole pyridine / acetonitrile mixed solution with a volume ratio of pyridine to acetonitrile of 3:5, and Cap2 being a 20 vol% acetic anhydride acetonitrile solution. The molar ratio of N-methylimidazole in the Cap1 capping reagent, acetic anhydride in the Cap2 capping reagent, and the nucleic acid sequence linked to the solid support was 1:1:1.

[0225] The oxidation reaction conditions were the same for each step. The oxidation reaction conditions were a temperature of 25°C, a reaction time of 3 seconds, an oxidation reagent concentration of 0.05 M iodine solution, a molar ratio of iodine to the nucleic acid sequence linked to the solid support in the coupling reaction of 30:1, and the oxidation reaction was carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine of 1:9). The sulfidation reaction conditions were a temperature of 25°C, a reaction time of 360 seconds, a thio reagent concentration of 0.2 M pyridine solution of xanthogen hydride, a molar ratio of thio reagent to the nucleic acid sequence linked to the solid support in the coupling reaction of 4:1, and the sulfidation reaction was carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine of 1:9).

[0226] After the ligation of the final nucleotide monomer was complete, the nucleic acid sequence ligated to the solid support was sequentially cleaved, deprotected, purified, and desalted, and then freeze-dried to obtain the sense strand.

[0227] The cleavage and deprotection conditions were as follows: The nucleotide sequence linked to the synthesized solid support was placed in 25% by mass ammonia water, reacted at 55°C for 16 hours at a dose of 0.5 ml / μmol of ammonia water, the solvent was removed, and the mixture was vacuum concentrated until dry. After treatment with ammonia water, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine hydrofluoric acid were added to remove the 2'-O-TBDMS protection in ribose.

[0228] The purification and desalting conditions involved completing the nucleic acid purification by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH=8.1) with a water / acetonitrile mixed solvent (volume ratio of water to acetonitrile 9:1), and eluent 2 was 1.5 M sodium chloride and 20 mM sodium phosphate (pH=8.1) with a water / acetonitrile mixed solvent (volume ratio of water to acetonitrile 9:1). The eluent ratio was eluent 1:eluent 2 = (100:0) to (50:50). The product eluents were collected and combined, and desalted using a reverse chromatography purification column. The desalting conditions included desalting using a dextran gel column with dextran gel G25 as the filler, and elution with deionized water.

[0229] Detection: Purity detection was performed using ion exchange chromatography (IEX-HPLC), and molecular weight detection was performed using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, it indicated that the 3'-end of the compound complexed with the siRNA sense strand was obtained.

[0230] (1.5.2) Synthesis of antisense strand AS The antisense strand was synthesized using a general solid-phase support. The reaction conditions for deprotection, coupling, capping, oxidation or sulfidation, the conditions for cleavage and deprotection, and the conditions for purification and desalting in the solid-phase synthesis method of the antisense strand were the same as those for the synthesis of the sense strand in step (1.5.1).

[0231] Detection: Purity detection was performed using ion exchange chromatography (IEX-HPLC), and molecular weight detection was performed using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, it indicated that the siRNA antisense strand was obtained.

[0232] (1.5.3) Synthesis of siRNA The sense strand synthesized in step (1.5.1) and the antisense strand synthesized in step (1.5.2) were mixed in an equimolar ratio, dissolved in water for injection, heated to 95°C, slowly cooled to room temperature, held at room temperature for 10 minutes, and a double-stranded structure was formed between the sense strand and the antisense strand through hydrogen bonding to obtain the target siRNA.

[0233] Detection: Each siRNA was diluted to a concentration of 0.2 mg / ml (calculated for siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ·cm (25℃)), and then molecular weight detection was performed using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometer, purchased from Waters, model number: LCT Premier). If the measured value matches the theoretical value, it indicates that the synthesized siRNA is the double-stranded nucleic acid sequence of the target design.

[0234] Preparation of double-stranded oligonucleotide (siRNA) conjugates Preparation Example 6 (1.6.1) Synthesis of sense chains Using a solid-phase synthesis method for phosphoramidite nucleic acids, the compounds linked to the solid support (i.e., CR01008Z, CR01013Z, L96-PS) were linked one nucleotide at a time in a circular manner following the nucleotide sequence, from 3' to 5' (compounds CR01008 and CR01013 can each be considered as one nucleotide monomer). Each linkage of nucleotide monomers involved four reaction steps: deprotection, coupling, capping, and oxidation or sulfidation. The reaction conditions for deprotection, coupling, capping, oxidation or sulfidation, cleavage and deprotection, purification and desalting during the synthesis of the sense strand in this example were the same as those for the synthesis of the sense strand in step (1.5.1) of Preparation Example.

[0235] (1.6.2) Synthesis of antisense chains Antisense chains were synthesized using a general-purpose solid-phase support. The reaction conditions for deprotection, coupling, capping, oxidation or sulfurization, cleavage and deprotection, purification and desalting in the solid-phase synthesis method of antisense chains were the same as those for step (1.5.2) of the antisense chain synthesis in Preparation Example 5.

[0236] Detection: Purity is detected using ion exchange chromatography (IEX-HPLC), and molecular weight is detected using a liquid chromatography-mass spectrometer (LC-MS, purchased from Waters, model number: LCT Premier). The measured molecular weight is compared with the theoretical value, and if the measured value matches the theoretical value, it indicates that an siRNA antisense strand has been obtained.

[0237] (1.6.3) Synthesis of siRNA The sense strand synthesized in step (1.6.1) and the antisense strand synthesized in step (1.6.2) were mixed in equimolar ratios, dissolved in sterile water for injection, heated to 95°C, slowly cooled to room temperature, and held at room temperature for 10 minutes to form a double-stranded structure between the sense strand and antisense strand via hydrogen bonding, thereby obtaining the target siRNA conjugate.

[0238] Detection: Each siRNA was diluted to a concentration of 0.2 mg / ml (calculated for siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ·cm (25℃)), and then molecular weight detection was performed using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometer, purchased from Waters, model number: LCT Premier). If the measured value matches the theoretical value, it indicates that the synthesized siRNA conjugate is the double-stranded nucleic acid sequence of the target design.

[0239] When the ligand was cluster 3CR01008, the structural formula of the siRNA conjugate was as follows:

[0240] [ka]

[0241] When the ligand was cluster CR01013, the structural formula of the siRNA conjugate was as follows:

[0242] [ka]

[0243] When the ligand was L96, the structural formula of the siRNA conjugate was as follows:

[0244] [ka]

[0245] however,

[0246] [ka] This represents siRNA.

[0247] The unmodified double-stranded oligonucleotide sequences relating to this disclosure are shown in Table 1.

[0248] [Table 4-1]

[0249] [Table 4-2]

[0250] [Table 4-3]

[0251] [Table 4-4]

[0252] [Table 4-5]

[0253] [Table 4-6]

[0254] The modified double-stranded oligonucleotide sequences relating to this disclosure are shown in Table 2.

[0255] [Table 5-1]

[0256] [Table 5-2]

[0257] [Table 5-3]

[0258] [Table 5-4]

[0259] [Table 5-5]

[0260] [Table 5-6]

[0261] [Table 5-7]

[0262] [Table 5-8]

[0263] [Table 5-9]

[0264] [Table 5-10]

[0265] [Table 5-11]

[0266] [Table 5-12]

[0267] [Table 5-13]

[0268] [Table 5-14]

[0269] In this disclosure, the meanings of base composition and modification were as follows: Uppercase A, U, G, C, and T represent the base composition of a nucleotide; lowercase m indicates that the nucleotide adjacent to the left of m is modified with a 2'-methoxy group; lowercase f indicates that the nucleotide adjacent to the left of f is modified with a 2'-fluoro group; lowercase d indicates that the nucleotide adjacent to the left of d is a deoxyribonucleotide; and lowercase s indicates that the two nucleotides adjacent to the left and right of s are linked by a thiophosphate bond.

[0270] The information that the ligand relating to this disclosure is a double-stranded oligonucleotide conjugate of L96 is shown in Table 3.

[0271] [Table 6-1]

[0272] [Table 6-2]

[0273] [Table 6-3]

[0274] [Table 6-4]

[0275] The information that the ligand relating to this disclosure is a double-stranded oligonucleotide conjugate of cluster 3 CR01008 is shown in Table 4.

[0276] [Table 7-1]

[0277] [Table 7-2]

[0278] [Table 7-3]

[0279] [Table 7-4]

[0280] [Table 7-5]

[0281] [Table 7-6]

[0282] Biological detection experiment Unless otherwise specified, the reagent materials (Table 5) and instrument devices (Table 6) used in this application are all derived from commercially available products from the following manufacturers.

[0283] [Table 8]

[0284] [Table 9]

[0285] Unless otherwise specified, the human hepatocellular carcinoma cell line Huh7 used in this disclosure was purchased from Wuhan Procell Life Technology Co., Ltd., the experimental animals C57BL / 6J mice used in this disclosure were purchased from Zhejiang Vitong Lihua Laboratory Animal Technology Co., Ltd., the experimental animals Balb / c mice used in this disclosure were purchased from Zhejiang Vitong Lihua Laboratory Animal Technology Co., Ltd., the BKS-DB mice used in this disclosure were purchased from GemPharmatech, and the triglyceride and total cholesterol levels in the serum of the BKS-DB mice used in this disclosure were detected by Anling Biomedical (Suzhou) Co., Ltd.

[0286] In the context of this disclosure, unless otherwise specified, the Real-time PCR detection data for the activity experiments related to this disclosure were all calculated using the ΔΔCt method to obtain relative quantification of target gene mRNA within each test group, and the general method of calculation is as follows. ΔCt(test group) = Ct(test group target gene) - Ct(test group reference gene) ΔCt(control group) = Ct(control group target gene) - Ct(control group reference gene) ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group mean) ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group mean) The mRNA expression levels of the target gene in the test group were normalized using the control group as a baseline, and the residual expression level of the target gene mRNA in the control group was defined as 100%. Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100% Suppression rate of target gene mRNA in the test group = 100% - relative expression level of target gene mRNA in the test group In the context of this disclosure, unless otherwise specified, all in vivo activity experimental data are expressed as X±STDEV, and all experimental data were graphed and analyzed using GraphPad prism 8.0 software.

[0287] Example 1: Evaluation of in vitro activity of siRNA In this example, the inhibitory activity of the target gene INHBE in cells of RX008001 to RX008154, which target the same INHBE site, was evaluated using a method for evaluating target gene suppression activity in the human hepatocellular carcinoma cell line Huh7, with RX000001 used as a negative control.

[0288] Sample preparation: After centrifuging each of the above siRNA samples, an appropriate amount of PBS was added according to the specifications of each tube to dissolve them and prepare a 20 μM mother liquor. The mother liquor was then serially diluted with PBS to 1 μM and 0.1 μM working solutions, or 0.1 μM and 0.01 μM working solutions, and dose tests were performed at final double-stranded cell concentrations of 10 nM and 1 nM, or 1 nM and 0.1 nM.

[0289] 96-well transfection and detection: Huh7 cells grown to a near-confluence state using trypsin were digested, the cells were washed, and a cell suspension was prepared. 100 μL of the cell suspension was placed in each well of a 96-well plate, resulting in 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. 24 hours after the cells had adhered to the plate wall, the DMEM medium was aspirated and removed from the 96-well plate. 80 μL of Opti-MEM® medium was added to each well, and the 96-well plate was placed in the incubator and cultured. 1 μL of 1 μM, 0.1 μM, or 0.01 μM working solution was dispersed in 9 μL of Opti-MEM to form an siRNA mixture. 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and uniformly mixed with the various siRNA mixtures to form a transfection complex. After incubating the transfection complex at room temperature for 10 minutes, the transfection complex was added to a 96-well plate at a concentration of 20 μL / well. After 4 hours of incubation, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator and incubated for 24 hours.

[0290] The 96-well plate was removed, and total RNA was extracted using a fully automated nucleic acid extraction system (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (GO-MNTR-100, also purchased from Zhejiang Hanwei Technology Co., Ltd.) following the standard procedure for total RNA extraction.

[0291] Using a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622), Oligo(dT) 18Reverse transcription primers were selected, and the reverse transcription reaction was performed by placing a 20 μL reverse transcription system according to the method described in the reverse transcription kit specifications. Subsequently, the expression level of target gene mRNA in HepG2 cells was detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad CFX Opus 384). In this real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the reference gene, and the target gene and the GAPDH reference gene were detected using primers targeting the target gene and primers targeting the GAPDH reference gene, respectively. The sequences of the detection primers are shown in Table 7.

[0292] [Table 10]

[0293] In the real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression levels of target gene mRNA within each test group using the ΔΔCt method according to the technical method described in the embodiment.

[0294] [Table 11-1]

[0295] [Table 11-2]

[0296] [Table 11-3]

[0297] [Table 12]

[0298] Example 2: Evaluation of the activity of an siRNA conjugate with L96 as a ligand in vivo in a high-pressure hydro-injection (HDI) mouse model.

[0299] This example evaluated the inhibitory activity against the target gene INHBE of a conjugate in which the L96 ligand was compounded at the 3' end of the siRNA sense strand of the same INHBE target site, using a Balb / c mouse hydro-powered injection model.

[0300] Plasmid construction: pcDNA-CMV-RG008 plasmid (ID: nM_031479.5), constructed by Sangon Biotech (Shanghai).

[0301] Building a mouse model: The Balb / c mouse hydrodynamic injection model was constructed by high-pressure, high-speed injection of a pcDNA-CMV-RG008 plasmid solution into the mouse body via the tail vein. On day 3 of the experiment, 10 μg of pcDNA-CMV-RG008 was injected into the mouse tail vein by hydrodynamics within 5 seconds, with an injection volume of 8% of the mouse's body weight. The plasmid DNA for injection was diluted with physiological saline, the solution was prepared before injection, and stored at 4°C.

[0302] The animals were divided into groups, administered the substance, and tissue samples were collected. Balb / c mice aged 6-8 weeks were randomly divided into groups based on body weight (all female), with 5 mice per group. Each test group was administered the planned dose of drug conjugate, and a PBS control group was added. The dose for all mice was calculated based on body weight and administered as a single subcutaneous injection in the abdomen. Each drug conjugate was administered in a PBS solution at a concentration of 0.1 mg / mL (calculated using siRNA), with a dosage volume of 10 mL / kg mouse body weight, meaning the dose of each drug conjugate was 1 mg / kg mouse body weight (calculated using siRNA). The PBS control group was administered the same volume of PBS solution (without drug conjugate). The day of administration was designated as day 0 (D0), plasmid injection was performed on day 3 (D3), and all 5 mice in all groups were euthanized on day 4 (D4). Each euthanized mouse was dissected macroscopically, and liver tissue was collected from each euthanized mouse. The liver tissue was then measured to approximately 2 mm. 3 It was cut into small chunks and stored in RNA later.

[0303] For each mouse, an appropriate amount of liver tissue sample was taken from the RNA later, the liver tissue sample was disrupted for 60 seconds using a Tissuelyser II fully automated tissue homogenizer, and total RNA was extracted using a fully automated nucleic acid extraction system (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) following the standard procedure for total RNA extraction.

[0304] For each mouse, 1 μg of total RNA was taken, and using a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622), Oligo(dT)18 reverse transcription primers were selected. A 20 μL reverse transcription system was then prepared according to the instructions in the reverse transcription kit specifications, and the reverse transcription reaction was carried out. After the reaction was complete, 60 μL of RNase-free water was added to the reverse transcription system to obtain the cDNA solution. Subsequently, the expression level of target gene mRNA in the animals was detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene in the plasmid backbone was used as the reference gene, and the target gene and the Nero reference gene were detected using primers targeting the target gene and primers targeting the Nero reference gene, respectively. See Table 10 for the sequences of the detection primers.

[0305] [Table 13]

[0306] According to the method described in the specification of the Real-Time Quantitative PCR Kit, 10 μL Real-Time PCR reaction systems were placed in each PCR detection well. Each reaction system contained 4 μL of cDNA solution obtained by the reverse transcription reaction described above, 5 μL of TaqMan® Fast Advanced Master Mix (2×), 0.15 μL of 10 μM upstream primer, 0.15 μL of 10 μM downstream primer, 0.15 μL of 10 μM probe primer, and 0.55 μL of RNase-Free H2O. The prepared reaction systems were placed in a Real-Time Quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-Time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and stretching at 60°C for 30 s, and the denaturation, annealing and stretching process was repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression levels of target gene mRNA within each test group using the ΔΔCt method according to the technical method described in the embodiment.

[0307] [Table 14]

[0308] According to the results in Figure 1 and Table 11, at a dose of 1 mg / kg, RZ008003, RZ008019, RZ008020, RZ008023, RZ008026, and RZ008028 significantly suppressed INHBE mRNA expression, with suppression rates greater than 70%.

[0309] Example 3: Evaluation of the activity of an siRNA conjugate using L96 as a ligand in C57BL / 6J mice. This example evaluated the inhibitory activity of the ligand L96 siRNA conjugates RZM08001-RZM08024 on mRNA in C57BL / 6J mouse liver tissue, with RZ000001 used as a negative control.

[0310] The animals were divided into groups, administered the substance, and tissue samples were collected. Six- to eight-week-old C57BL / 6J male mice (Weitong Lihua Experimental Animal Technology Co., Ltd., Zhejiang Province) were randomly divided into groups of five mice each based on body weight. Each test group was administered the planned dose of drug conjugate, and a PBS control group was added. The dose for all mice was calculated based on body weight, with a dosage volume of 10 mL / kg mouse body weight. A single dose was administered by subcutaneous abdominal injection, with each drug conjugate administered in a PBS solution at a concentration of 0.3 mg / mL (calculated using siRNA), meaning the dose of each drug conjugate was 3 mg / kg mouse body weight (calculated using siRNA). The PBS control group was administered the same volume of PBS solution (without drug conjugate).

[0311] The day of administration was designated as day 0 (D0), and on day 7 after administration (D7), all groups of mice were euthanized. Each euthanized mouse was dissected macroscopically, and liver tissue was collected. The liver tissue was then measured to approximately 2 mm. 3 The DNA was cut into small chunks, stored in RNA later, and the INHBE mRNA expression level was measured. In the real-time fluorescence quantitative PCR method, the GAPDH gene was used as the reference gene, and detection was performed using primers targeting the target gene and primers targeting the GAPDH reference gene, respectively. See Table 12 for the sequences of the detection primers.

[0312] [Table 15]

[0313] In the real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression levels of target gene mRNA within each test group using the ΔΔCt method according to the technical method described in the embodiment.

[0314] [Table 16]

[0315] As shown in Figure 2 and Table 13, RZM08015 and RZM08019 significantly reduced mRNA expression levels in C57BL / 6J mouse liver tissue on day 7 after a single subcutaneous administration of 3 mg / kg, with the inhibitory effect of RZM08019 mRNA reaching 70%.

[0316] Example 4: Evaluation of the in vivo activity of an siRNA conjugate with ligand L96 in C57BL / 6J mice. This example evaluated the mRNA suppression activity of RZM08019 in C57BL / 6J mouse liver tissue after single-dose administration of different doses of L96 carrier conjugate.

[0317] The animals were divided into groups, administered the substance, and tissue samples were collected. Six- to eight-week-old C57BL / 6J male mice (Weitong Lihua Experimental Animal Technology Co., Ltd., Zhejiang Province) were randomly divided into groups of five mice each based on body weight. Each test group was administered the planned dose of drug conjugate, and a PBS control group was added. The dose for all mice was calculated based on body weight, with a dosage volume of 10 mL / kg mouse body weight. A single dose was administered by subcutaneous abdominal injection, with each drug conjugate administered in PBS solution at concentrations of 0.9 mg / mL, 0.6 mg / mL, 0.3 mg / mL, 0.1 mg / mL, and 0.03 mg / mL (calculated using siRNA), i.e., the doses for each drug conjugate were 9 mg / kg, 6 mg / kg, 3 mg / kg, 1 mg / kg, and 0.3 mg / kg mouse body weight (calculated using siRNA). The PBS control group was administered the same volume of PBS solution (without drug conjugate).

[0318] The day of administration was designated as day 0 (D0), and on day 7 after administration (D7), all groups of mice were euthanized. Each euthanized mouse was dissected macroscopically, and liver tissue was collected. The liver tissue was then measured to approximately 2 mm. 3The DNA was cut into small chunks, stored in RNA later, and the INHBE mRNA expression level was measured. In the real-time fluorescence quantitative PCR method, the GAPDH gene was used as the reference gene, and the target gene and the GAPDH reference gene were detected using primers targeting the target gene and primers targeting the GAPDH reference gene, respectively. See Table 12 for the sequences of the detection primers.

[0319] In the real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression level and repression rate of target gene mRNA within each test group using the ΔΔCt method according to the technical method described in the embodiment.

[0320] [Table 17]

[0321] According to the data in Table 14 and Figure 3, RZM08019 was found to be able to dose-dependently reduce mRNA expression levels in C57BL / 6J mouse liver tissue.

[0322] Example 5: Efficacy evaluation of siRNA conjugate RZM08019 in BKS-DB mice in vivo In this example, the inhibitory activity of RZM08019 against the target gene INHBE in mice was evaluated using a mouse in vivo target gene suppression activity evaluation method, and the regulatory effect of RZM08019 on blood lipids in mice was evaluated by detecting changes in triglycerides and total cholesterol in mouse serum using an automated biochemical analyzer.

[0323] The animals were divided into groups, administered the substance, and tissue samples were collected. Male BKS-DB mice (GemPharmatech) aged 6-8 weeks were randomly divided into groups according to body weight. Six mice were assigned to the PBS group, and six mice were assigned to the drug conjugate RZM08019 Q2W x 2 group (administered once every two weeks for a total of two doses). The dosage for all mice was calculated based on body weight, with a dosage volume of 10 mL / kg mouse body weight. RZM08019 was repeatedly administered by subcutaneous abdominal injection, once every two weeks for a total of two doses. The drug conjugate was administered in a PBS solution at a concentration of 0.9 mg / mL (calculated using siRNA), i.e., the dosage was 9 mg / kg mouse body weight (calculated using siRNA). The PBS control group was administered the same volume of PBS solution (without the drug conjugate). The day of administration was designated as day 0 (D0), and serum samples were collected from all groups of mice on days 7, 14 (D14), 21 (D21), 28 (D28), and 35 (D35) after administration. These samples were sent to Anling Biomedical (Suzhou) Co., Ltd., where triglycerides and total cholesterol in the serum were detected using a fully automated biochemical analyzer.

[0324] On day 35 after administration (indicated as D35), all groups of mice were euthanized. Each euthanized mouse was dissected macroscopically, and liver tissue was collected. The liver tissue was then measured to approximately 2 mm. 3 The DNA was cut into small chunks, stored in RNA later, and the INHBE mRNA expression level was measured. In the real-time fluorescence quantitative PCR method, the GAPDH gene was used as the reference gene, and the target gene and the GAPDH reference gene were detected using primers targeting the target gene and primers targeting the GAPDH reference gene, respectively. See Table 12 for the sequences of the detection primers.

[0325] In the real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression level and repression rate of target gene mRNA within each test group using the ΔΔCt method according to the technical method described in the embodiment.

[0326] [Table 18]

[0327] [Table 19]

[0328] [Table 20]

[0329] According to the data results in Tables 15-17 and Figures 4-5, after subcutaneous injection of RZM08019 9 mg / kg Q2W×2 conjugate into BKS-DB mice, RZM08019 was able to significantly reduce the CHO expression level in mouse serum. On day 35 of the study, a significant reduction in mRNA expression level in mouse liver tissue was observed, indicating that the inhibitory effect was greater than 85%.

[0330] Example 6: Evaluation of the in vitro activity of an siRNA conjugate using CR01008 x 3 as a ligand. In this example, the intracellular inhibitory activity of the CR01008-siRNA conjugate against the target gene INHBE was evaluated using a method for evaluating target gene repression activity in the human hepatocellular carcinoma cell line Huh7.

[0331] Sample preparation: After centrifuging each of the above siRNA samples, an appropriate amount of PBS was added according to the specifications of each tube to dissolve them and prepare a 20 μM mother liquor. The mother liquor was then serially diluted with PBS to a 0.1 μM working solution, and dose tests were performed at a final double-stranded siRNA concentration of 1 nM.

[0332] 96-well transfection and detection: Huh7 cells grown to a near-confluence state using trypsin were digested, and the cells were washed to prepare a cell suspension. 100 μL of the cell suspension was placed in each well of a 96-well plate, resulting in 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. 24 hours after the cells had adhered to the plate wall, the DMEM medium was aspirated and removed from the 96-well plate. 80 μL of Opti-MEM® medium was added to each well. The 96-well plate was then placed in the incubator and cultured. 1 μL of 0.1 μM working solution was dispersed in 9 μL of Opti-MEM to form an siRNA mixture, and 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and uniformly mixed with the various siRNA mixtures to form a transfection complex. After incubating the transfection complex at room temperature for 10 minutes, the transfection complex was added to the 96-well plate at a volume of 20 μL / well. After 4 hours of incubation, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator and incubated for 24 hours.

[0333] The 96-well plate was removed, and total RNA was extracted using a fully automated nucleic acid extraction system (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (GO-MNTR-100, also purchased from Zhejiang Hanwei Technology Co., Ltd.) following the standard procedure for total RNA extraction.

[0334] Using a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622), Oligo(dT) 18Reverse transcription primers were selected, and the reverse transcription reaction was performed by placing a 20 μL reverse transcription system according to the method described in the reverse transcription kit specifications. Subsequently, the expression level of target gene mRNA in HepG2 cells was detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad CFX Opus 384). In this real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the reference gene, and the target gene and the GAPDH reference gene were detected using primers targeting the target gene and primers targeting the GAPDH reference gene, respectively. The sequences of the detection primers are shown in Table 7.

[0335] In the real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression level and repression rate of target gene mRNA within each test group using the ΔΔCt method according to the technical method described in the embodiment.

[0336] [Table 21]

[0337] According to the data results in Table 18 and Figure 6, it was found that the siRNA conjugate according to this example can significantly suppress the INHBE mRNA expression level in Huh7 cells.

[0338] Finally, it should be noted that the above embodiments are merely for illustrating, and not limiting, the present invention. Although the present invention has been described in detail with reference to the embodiments described above, it is still possible to modify the invention described in the embodiments described above, or to substitute some or all of its technical features. Those skilled in the art should understand that such modifications or substitutions do not cause the essence of the corresponding invention to deviate from the scope of the invention in each embodiment of the present invention.

Claims

1. A double-stranded oligonucleotide targeting the INHBE gene, comprising a sense strand and an antisense strand, wherein the antisense strand and the sense strand are complementary or nearly complementary, where nearly complementary means that there are three or fewer nucleotide mismatches in the double-stranded regions of the sense strand and the antisense strand, and the sense strand comprises a nucleotide sequence that is the same as or nearly the same as at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence, where nearly the same means that there are three or fewer nucleotide differences between the sense strand and at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence.

2. The double-stranded oligonucleotide according to claim 1, characterized in that the sense strand includes a nucleotide sequence in which the difference between at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence is two or fewer nucleotides, and preferably the difference between nucleotides is one or fewer.

3. The double-stranded oligonucleotide according to claim 1, characterized in that the antisense strand and at least 15, 16, 17, 18, 19, 20, 21, 22, and 23 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 309 are complementary or nearly complementary, wherein nearly complementary means that there are three or fewer nucleotide mismatches in the complementary region.

4. It comprises one sense strand and one antisense strand, wherein the antisense strand contains a nucleotide sequence in which at least 15 consecutive nucleotides are in any of the sequences indicated by SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence in which the difference from the said at least 15 consecutive nucleotides is 3 or less. The sense strand includes a nucleotide sequence that forms a double-stranded region at least partially inversely complementary or nearly complementary to the antisense strand, wherein nearly complementary means that there are three or fewer nucleotide mismatches in the double-stranded regions of the sense strand and the antisense strand. The double-stranded oligonucleotide according to claim 1.

5. The antisense strand includes a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from any of the nucleotide sequences indicated by SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is 3 or less. Preferably, the antisense strand includes a nucleotide sequence in which at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from any of the nucleotide sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is two or less. Preferably, the antisense strand includes a nucleotide sequence in which at least 19, at least 20, or at least 21 consecutive nucleotides from any of the nucleotide sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence in which the difference between such consecutive nucleotides is one or less. Preferably, the antisense strand is selected from or includes any of the nucleotide sequences shown in SEQ ID NO. 155 to SEQ ID NO. 308 in Table 1. Preferably, in the 5'→3' direction, the 2nd to 19th positions of the antisense strand include at least 15 nucleotides from the 2nd to 19th positions of any nucleotide sequence shown in Table 1 as SEQ ID NO. 155 to SEQ ID NO. 308, or a nucleotide sequence in which the difference from the at least 15 nucleotides is less than 3, as described in claim 4.

6. The sense strand includes a nucleotide sequence in which at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides from any of the nucleotide sequences indicated by SEQ ID NO. 1 to SEQ ID NO. 154 in Table 1, or a nucleotide sequence in which the difference between the consecutive nucleotides is 3 or less. Preferably, the sense strand contains at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides from any of the nucleotide sequences shown in SEQ ID NO. 1 to SEQ ID NO. 154 in Table 1. Preferably, the sense strand is selected from or includes any nucleotide sequence shown in Table 1 as SEQ ID NO. 1 to SEQ ID NO. 154, characterized in that the double-stranded oligonucleotide according to claim 4.

7. Each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides. Preferably, almost all nucleotides of the sense strand or the antisense strand are selected from modified nucleotides. Preferably, all nucleotides of the sense strand and the antisense strand are selected from modified nucleotides, as described in claim 4.

8. The modified nucleotides are each independently 2'-halogen, 2'-deoxy, and 2'-O-(CH ２ ) ｎ -R １ A nucleotide modified with or selected from nucleotide analogs, wherein the nucleotide analog is one or more selected from PNA, MNA, BNA, LNA, GNA, TNA, and UNA. n is selected from 0, 1 or 2, and R １ is an optionally substituted C １～６ alkyl group, an optionally substituted C １～６ alkoxy group or -Si(R １ａ ) ３ selected from, each R １ａ is independently an optionally substituted C １～６ alkyl group or an optionally substituted C １～６ alkoxy group selected from, Preferably, 2'-O-(CH ２ ) ｎ -R １ is 2'-O-CH ３ , 2'-O-CH ２ -O-CH ３ , 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH ２ -O-CH ２ -CH ３ , 2'-O-CH ２ -O-CH ２ -CF ３ or 2'-O-CH ２ -CH ２ -O-CH ３ A double-stranded oligonucleotide according to claim 7, selected from the above.

9. The sense strand or the antisense strand includes a 3' overhang having at least one nucleotide, Preferably, the antisense strand includes a 3' overhang having at least one nucleotide. Preferably, the sense strand or the antisense strand includes a 3' overhang having at least two nucleotides. Preferably, the antisense strand includes a 3' overhang having at least two nucleotides. Preferably, the antisense strand includes a 3' overhang having two nucleotides. Preferably, the sense chain and / or the antisense chain independently contain one or more thiophosphate bonds. Preferably, the sense strand includes two consecutive thiophosphate bonds between the terminal nucleotides at the 5' end. Preferably, the double-stranded oligonucleotide according to claim 4, wherein the antisense strand contains two consecutive thiophosphate bonds between the 3' and 5' terminal nucleotides, respectively.

10. All nucleotides of the sense strand and all nucleotides of the antisense strand are selected from modified nucleotides, and the double-stranded region formed by the sense strand and the antisense strand is as shown in the following formula (I): SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(I), However, SS represents the sense chain, and AS represents the antisense chain. Each N is independently selected from nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, or nucleotides modified with 2'-deoxy. Each X is independently 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, and 2'-O-CH ２ -O-CH ２ -CH ３ , 2'-O-CH ２ -O-CH ２ -CF ３ , 2'-O-CH ２ -CH ２ -O-CH ３ Selected from, The above a, a', p, p', b, b', q, q', c, c', r', and d' each independently represent the number of nucleotides, where a' is selected from integers between 3 and 8, p' from integers between 0 and 3, b' from integers between 4 and 13, q' from integers between 0 and 4, c' from integers between 3 and 9, r' from integers between 0 and 3, d' from integers between 0 and 9, a is selected from integers between 4 and 7, p is selected from integers between 0 and 1, b is selected from integers between 4 and 8, q is selected from integers between 0 and 4, and c is selected from integers between 6 and 10, and p', q', r', p, and q cannot be 0 at the same time, and 0 ≤ q' + r' ≤ 4. Preferably, a' is selected from integers 3 to 8, p' is selected from 0 or 1, b' is selected from integers 4 to 13, q' is selected from 0 or 1, c' is selected from integers 3 to 9, r' is selected from 0 or 1, d' is selected from integers 1 to 8, a is selected from integers 4 to 7, p is selected from 1, b is selected from integers 4 to 8, q is selected from 0 or 1, and c is selected from integers 6 to 10. Preferably, each N is independently selected from nucleotides modified with 2'-O-methyl and nucleotides modified with 2'-fluoro. Preferably, each X is independently a nucleotide modified with 2'-O-methyl, a nucleotide modified with 2'-O-MOE (methoxyethyl), a nucleotide modified with 2'-O-TBDMS, a nucleotide modified with 2'-O-TIPS, a nucleotide modified with 2'-O-TOM, or a nucleotide modified with 2'-O-CH ２ -O-CH ２ -CH ３ Modified nucleotides, 2'-O-CH ２ -O-CH ２ -CF ３ Selected from nucleotides modified with Preferably, each X is independently a nucleotide modified with 2'-O-methyl, a nucleotide modified with 2'-O-MOE, or 2'-O-CH ２ -O-CH ２ -CH ３ Modified nucleotides, 2'-O-CH ２ -O-CH ２ -CF ３ Selected from nucleotides modified with Preferably, the double-stranded oligonucleotide according to any one of claims 1 to 9, wherein the double-stranded region comprises at least one nucleotide modified with 2'-O-methoxyethyl or nucleotide modified with 2'-O-ethoxymethyl.

11. In the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense chain are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl or nucleotides modified with 2'-O-methoxyethyl. Preferably, in the direction from the 5' end to the 3' end, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense chain are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl or nucleotides modified with 2'-O-methoxyethyl. Preferably, in the direction from the 5' end to the 3' end, any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense chain are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl or nucleotides modified with 2'-O-methoxyethyl. Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense chain are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl or nucleotides modified with 2'-O-methoxyethyl. Preferably, the antisense chain contains at least one nucleotide modified with 2'-O-methoxyethyl. Preferably, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from a nucleotide modified with 2'-O-methoxyethyl, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl. Preferably, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from a nucleotide modified with 2'-O-methoxyethyl, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl. Preferably, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from nucleotides modified with 2'-O-methoxyethyl, any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl. Preferably, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from nucleotides modified with 2'-O-methoxyethyl, the nucleotides at positions 2, 6, 9, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl. Preferably, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from nucleotides modified with 2'-O-methoxyethyl, the nucleotides at positions 2, 6, 12, 14, and 16 are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl. The double-stranded oligonucleotide according to claim 10.

12. In the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7 to 10 of the sense strand are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl. Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the sense strand are selected from nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-methyl. The double-stranded oligonucleotide according to claim 10.

13. The antisense strand includes a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from any modified antisense strand nucleotide sequence shown in Table 2, or a nucleotide sequence in which the difference between such consecutive nucleotides is 3 or less. Preferably, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from the nucleotide sequence of any modified antisense strand shown in Table 2. Preferably, the antisense strand is selected from or comprises any modified antisense strand nucleotide sequences shown in Table 2, as described in claim 10.

14. The sense strand includes a nucleotide sequence in which at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides from any modified sense strand nucleotide sequence shown in Table 2, or a nucleotide sequence in which the difference between such consecutive nucleotides is one, two, or three nucleotides. Preferably, the sense strand of the double-stranded oligonucleotide is selected from or includes any modified sense strand nucleotide sequences shown in Table 2, as described in claim 10.

15. The double-stranded oligonucleotide according to claim 10, characterized in that the sense strand of the double-stranded oligonucleotide is selected from or contains any modified sense strand nucleotide sequence shown in Table 2, and the antisense strand is selected from or contains any modified antisense strand nucleotide sequence shown in Table 2.

16. The aforementioned double-stranded oligonucleotide is selected from siRNA. A double-stranded oligonucleotide according to any one of claims 1 to 15.

17. A double-stranded oligonucleotide and a ligand capable of binding to one or more cell receptors, according to any one of claims 1 to 15, Preferably, the ligand is compounded with the sense chain and / or the antisense chain. Preferably, the ligand is compounded to the 3' and / or 5' ends of the sense chain. Preferably, the ligand is compounded at the 3' end of the sense chain. Preferably, the cell receptor is selected from asialoclycoprotein receptors. Preferably, the ligand comprises galactose or a galactose cluster, characterized in that it is a double-stranded oligonucleotide conjugate.

18. The ligand is selected from the structure represented by formula (101), its isomers, or pharmaceutically acceptable salts thereof. 【Chemistry 1】 however, ＊ This represents a composite site between the ligand and the sense chain or the antisense chain, m is selected from 1, 2, 3, or 4. Each Z is independently selected from either a hydroxyl group or a mercapto group. Each p is independently selected from 1, 2, or 3. Each q is independently selected from 1, 2, or 3. Each R is independently selected from H, an optionally substituted C1-C6 alkyl group, or an optionally substituted C1-C6 alkoxy group. Each L is an independently and arbitrarily substituted C2-C20 alkylene group or 【Chemistry 2】 Selected from, R Ｌａ and R Ｌｂ k is independently and arbitrarily selected from C1 to C10 alkylene groups, and k is selected from 1, 2, 3, 4 or 5. Each Y is independently selected from O, S, or NH. Preferably, m is selected from 1, 2, or 3. Preferably, Z is selected from hydroxyl groups. Preferably, p is selected from 1. Preferably, q is selected from 1. Preferably, R is selected from H, Preferably, each L independently comprises a C1-C10 alkylene group or 【Transformation 3】 Selected from, however, R Ｌａ and R Ｌｂ The group is independently selected from C1 to C5 alkylene groups, and k is 1, 2, or 3. Preferably, k is selected from 1. Preferably, each L is independently 【Chemistry 4】 Selected from, Preferably, Y is selected from O, characterized in that the conjugate according to claim 17.

19. The ligand is selected from the following structures or their isomers or pharmaceutically acceptable salts: 【Transformation 5】 The conjugate according to feature 17.

20. The ligand is selected from the structures shown below, their isomers, or pharmaceutically acceptable salts thereof. 【Transformation 6】 however, ＊ The conjugate according to claim 17, characterized in that represents a composite site of the ligand and the sense chain or the antisense chain.

21. (I) A double-stranded oligonucleotide according to any one of claims 1 to 16, and / or (II) A composition comprising any one of the conjugates described in any one of claims 17 to 20.

22. (I) A double-stranded oligonucleotide according to any one of claims 1 to 16, and / or (II) The conjugate according to any one of claims 17 to 20, and / or (III) Use of the composition according to claim 21 in the preparation of a drug for preventing and / or treating a disease or condition mediated by any of the INHBE genes, Preferably, the disease or condition described above includes, but is not limited to, metabolic disorders, type 2 diabetes, obesity, elevated triglyceride levels, fatty malnutrition, hepatitis, fatty liver disease, hypercholesterolemia, elevated liver enzymes, non-alcoholic steatohepatitis (NASH), cardiovascular disease, cardiomyopathy, hypertension, and / or heart failure, in addition to the risk of having or progressing these conditions.

23. (I) A double-stranded oligonucleotide according to any one of claims 1 to 16, and / or (II) The conjugate according to any one of claims 17 to 20, and / or (III) A pharmaceutical composition comprising any of the compositions described in claim 21 and a pharmaceutically acceptable auxiliary or adjuvant.

24. (I) A double-stranded oligonucleotide according to any one of claims 1 to 16, and / or (II) The conjugate according to any one of claims 17 to 20, and / or (III) The composition according to claim 21, and / or (IV) A method for reducing the expression or activity of the INHBE gene, characterized by bringing any of the pharmaceutical compositions described in claim 23 into contact with a cell.

25. (I) A double-stranded oligonucleotide according to any one of claims 1 to 16, and / or (II) The conjugate according to any one of claims 17 to 20, and / or (III) The composition according to claim 21, and / or (IV) A method for preventing and / or treating a disease or condition mediated by the INHBE gene, comprising administering to a subject in a pharmaceutically acceptable amount any of the pharmaceutical compositions described in claim 23.

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